Chapter 16 Somato-dendritic processing of postsynaptic potentials Iii Chapter 16 Somato-dendritic processing of postsynaptic potentials Iii. Role of high-voltage-activated depolarizing currents From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved.

From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.1 Examples of neurons of the mammalian central nervous system from which dendritic spikes are recorded. Drawing of neurons (left) with the corresponding recording of dendritic spikes (right) evoked by afferent stimulation. (a) Pyramidal neuron of the neocortex. (b) Pyramidal neuron of the hippocampus. (c) Dopaminergic neurons of the substantia nigra. (d) Purkinje cell of the cerebellar cortex. Part (a) adapted from Seamans JK, Gorelova N, Yang CR (1997) Contribution of voltage-gated Ca2+ channels in the proximal versus distal dendrites to synaptic integration in prefrontal cortical neurons. J. Neurosci.17, 5936–5948, with permission. Part (b) drawing by Taras Pankevitch and Roustem Khazipov and adapted from Tsubokawa H, Ross WN (1996) IPSPs modulate spike backpropagation and associated [Ca2+]i changes in dendrites of hippocampal CA1 pyramidal neurons. J. Neurophysiol.76, 2896–2906, with permission. Part (c) drawing by Jérôme Yelnik and adapted from Häusser M, Stuart G, Racca C, Sakmann B (1995) Axonal initiation and active dendrite propagation of action potentials in substantia nigra neurons. Neuron15, 637–647, with permission. Part (d) adapted from Callaway JC, Lasser-Ross N, Ross WN (1995) IPSPs strongly inhibit climbing fiber-activated [Ca2+]i increases in the dendrites of cerebellar Purkinje neurons. J. Neurosci.15, 2777–2787, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved.

Figure 16.2 Characteristics of dendritic Na+ channels in a pyramidal neuron of the hippocampus. (a) Consecutive sweeps showing Na+ channel openings (dendrite-attached configuration, voltage clamp mode) in response to step depolarizations to −40 mV (VH = −70 mV). Most of the channel openings occur at the beginning of the step but there are some late reopenings. (b) Current–voltage plot of Na+ channel activity. Unitary current amplitude from a total of 27 patches is plotted as a function of membrane potential. Bars are standard error of the mean (SEM). The slope indicates a unitary conductance γ of 15 pS and the extrapolated reversal potential Erev is +54 mV. (c) Dendritic Na+ channel steady-state activation and inactivation characteristics. Activation is tested by applying depolarizing steps to −65 to 0 mV from VH = −90 mV. Inactivation is tested by applying a depolarizing step to −5 mV from a VH varying from −105 to −45 mV. The representative steady-state activation (black circle) and inactivation (open circle) plots for dendritic Na+ channels indicate that they are half-activated at V1/2 = −30 mV and half-inactivated at V1/2 = −62 mV. Adapted from Magee JC, Johnston D (1995) Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons. J. Physiol. (Lond.)487, 67–90, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 3

From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.2 Characteristics of dendritic Na+ channels in a pyramidal neuron of the hippocampus.(cont.) (a) Consecutive sweeps showing Na+ channel openings (dendrite-attached configuration, voltage clamp mode) in response to step depolarizations to −40 mV (VH = −70 mV). Most of the channel openings occur at the beginning of the step but there are some late reopenings. (b) Current–voltage plot of Na+ channel activity. Unitary current amplitude from a total of 27 patches is plotted as a function of membrane potential. Bars are standard error of the mean (SEM). The slope indicates a unitary conductance γ of 15 pS and the extrapolated reversal potential Erev is +54 mV. (c) Dendritic Na+ channel steady-state activation and inactivation characteristics. Activation is tested by applying depolarizing steps to −65 to 0 mV from VH = −90 mV. Inactivation is tested by applying a depolarizing step to −5 mV from a VH varying from −105 to −45 mV. The representative steady-state activation (black circle) and inactivation (open circle) plots for dendritic Na+ channels indicate that they are half-activated at V1/2 = −30 mV and half-inactivated at V1/2 = −62 mV. Adapted from Magee JC, Johnston D (1995) Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons. J. Physiol. (Lond.)487, 67–90, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved.

5 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.3 TTX-sensitive inward currents in dendrites of pyramidal neurons of the neocortex and Purkinje cells of the cerebellar cortex. (a) Neocortex. Rapidly inactivating inward current evoked by a depolarizing step to −10 mV (VH = −90 mV) in an outside-out dendritic macropatch excised from the apical dendrite of a layer V pyramidal neuron (439 μm from the soma) (control). This current is reversibly blocked in the presence of 500 nM of TTX in the external solution. (b) Purkinje cells. Voltage-activated currents evoked by a depolarizing step to −10 mV (VH = −120 mV) in outside-out macropatches excised from either the soma (left) or dendrite (right, 94 μm from the soma) of Purkinje cells using similar-sized patch pipettes. A rapidly inactivating inward current followed by an outward current that is more prominent in the somatic membrane are recorded (control). Rapidly inactivating currents in both somatic and dendritic patches are reversibly blocked by the presence of 500 nM of TTX in the extracellular medium (TTX). Part (a) adapted from Stuart G, Sakmann B (1994) Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature367, 69–72, with permission. Part (b) adapted from Stuart GJ, Häusser M (1994) Initiation and spread of sodium action potentials in cerebellar Purkinje cells. Neuron13, 703–712, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 5

Figure 16.4 Schematic drawings of two hypotheses concerning the site of dendritic Na+ spike initiation. (a) In response to dendritic EPSPs, Na+ action potential (AP) is locally initiated in dendrites (black point) and then actively propagates to the soma-initial segment and along the axon. (b) In response to dendritic EPSPs, Na+ action potential is first initiated at the soma-initial segment (black point) and then actively propagates along the axon and backpropagates (actively or passively) into the dendritic tree. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 6

7 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.5 Site of initiation of Na+ action potential and its active backpropagation into the dendritic tree of pyramidal neurons of the neocortex. (a) Na+ action potential evoked by distal synaptic stimulation in layer I and simultaneously recorded from the soma and a dendrite (dendritic recording is 525 μm from the soma). (b) Comparison of active and passive propagation of Na+ action potential in the apical dendrite studied with simultaneous somatic and dendritic recordings. (b1) An action potential is evoked in the soma by a depolarizing current pulse (200 pA, soma). It propagates in the apical dendrite where it is recorded (dendrite, 310 μm from the soma). (b2) A simulated action potential waveform is injected in the soma in the presence of 1 μM of TTX. The somatic voltage response (soma) propagates passively in the dendrites where it is recorded at the same location as in b1 but in the presence of TTX (dendrite). The simulated somatic action potential is recorded later at the soma with a second somatic recording pipette (soma). (b3) Histogram of the average amplitude of dendritic action potentials recorded as in b1 (open column) and of dendritic responses recorded as in b2 (black column). Data are expressed as a percentage of the response recorded at the soma ± SEM; dendritic recordings 165–470 μm from the soma. Adapted from Stuart G, Sakmann B (1994) Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature367, 69–72, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 7

8 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.6 Site of initiation of Na+ action potential and its active backpropagation in the dendritic tree of dopaminergic neurons of the substantia nigra. (a) Spontaneous Na+ action potential recorded simultaneously at the soma and dendrite (top) and the morphological reconstruction of the filled recorded neuron (below) with the location of the somatic and dendritic pipettes. The axon origin is indicated. The action potential is observed to occur first at the dendritic recording site, 195 μm from the soma; the axon of this cell emerges from the dendrite from which the dendritic recording is made (215 μm from the soma). (b) (b1) An action potential is evoked in the soma by a depolarizing current pulse (200 pA, soma). It propagates in the dendrite where it is recorded (dendrite, 100 μm from the soma). (b2) A simulated action potential waveform is injected in the soma in the presence of 1 μM of TTX. The somatic voltage response (soma) propagates passively in the dendrites where it is recorded at the same location as in b1 but in the presence of TTX (dendrite). The simulated somatic action potential is recorded later at the soma with a second somatic recording pipette (soma). Adapted from Häusser M, Stuart G, Eacca C, Sakmann B (1995) Axonal initiation and active dendritic propagation of action potentials in substantia nigra neurons. Neuron15, 637–647, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 8

Figure 16.7 Initiation of action potential bursts is impaired by blockade of axosomatic Nav channels but not dendritic Nav channels. Schematic drawings in (a) and (b) show the site of pressure application of glutamate at dendritic sites (green pipette) and that of TTX (red pipette) next to the site of dendritic glutamate application in (a) or close to the axon hillock (red arrow) in (b). The somatic recording pipette is white. Scale bar: 50 μm. (a) Application of TTX (red) 1 s prior to the dendritic pulsed application of glutamate (green) has little effect (right) on evoked activity compared to that evoked under control conditions (left). (b) In contrast, axo-somatic application of TTX reduces the intensity of burst firing evoked by dendritic application of glutamate (right) compared to that evoked under control conditions (left). TTX is applied 1 s prior to glutamate application. From Blythe SN, Wokosin D, Atherton JF, Bevan MD (2009) Cellular mechanisms underlying burst firing in substantia nigra dopamine neurons. J. Neurosci.29, 15531–15541, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 9

10 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.8 Passive propagation of Na+ action potentials in the dendritic tree of Purkinje cells. (a) Simultaneous recordings at the soma and dendrite (108 μm from the soma) of a train of Na+ action potentials evoked by a somatic long depolarizing current pulse (100 pA). (b) (b1) An action potential is evoked in the soma by a depolarizing current pulse (soma). It propagates in the dendrite where it is recorded (dendrite, 47 μm from the soma). (b2) A simulated action potential waveform is injected in the soma in the presence of 1 μM of TTX. The somatic voltage response propagates passively in the dendrites where it is recorded at the same location as in b1 but in the presence of TTX (dendrite). The simulated somatic action potential is recorded later at the soma with a second somatic recording pipette (soma). Adapted from Stuart GJ, Häusser M (1994) Initiation and spread of sodium action potentials in cerebellar Purkinje cells. Neuron13, 703–712, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 10

11 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.9 P-type Ca2+ channel current in dendrites of Purkinje cells. (a) In the presence of 10 μM of ω-CgTx added to the 10 m M of Ba2+ pipette solution, currents are evoked in an outside-out macropatch of dendritic membrane by a depolarizing voltage ramp from −80 to +80 mV. The I/V plot shows that the evoked inward current peaks at −9 mV and activates at −44 mV. (b) Currents carried by 20 m M of Ba2+ evoked by voltage ramps (from −80 to +80 mV) in dendrite-attached macropatches in different conditions (left). Top: Averaged current in control conditions. Lower traces: Funnel web toxin (FTX) is first applied in the extracellular medium, then the patch is performed. Three different dendrite-attached patch recordings are shown (the approximate positions of the recording pipettes are indicated). The averaged currents recorded show the absence of inward Ba2+ current (right). Adapted from Usowicz MM, Sugimori M, Cherksey B, Llinas R (1992) P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells. Neuron9, 1185–1199, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 11

12 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.10 P-type Ca2+ current activated by climbing fiber EPSP in dendrites of Purkinje cells. (a) Intradendritic recording of the synaptically evoked climbing fiber response that is surmounted by two Ca2+ spikes (intracellular recording in current clamp mode). (b) Intradendritic recording at resting potential of the climbing fiber EPSP showing a 2–3 ms-wide Ca2+ spike and of the climbing fiber EPSP recorded during a concomitant IPSP (CF EPSP) (intracellular recording in current clamp mode). (c) Time course of [Ca2+]i recorded at a dendritic (d, top trace) and somatic (s, middle trace) site during spontaneous climbing fiber responses (s, bottom trace) recorded with simultaneous microfluorometric measurements of cytosolic free calcium concentration and intracellular (intrasomatic) electrophysiological recordings (current clamp mode). Part (a) adapted from Llinas R, Sugimori M (1980) Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. J. Physiol. (Lond.)305, 197–213, with permission. Part (b) adapted from Callaway JC, Lasser-Ross N, Ross WN (1995) IPSPs strongly inhibit climbing fiber-activated [Ca2+]i increases in the dendrites of cerebellar Purkinje neurons. J. Neurosci.15, 2777–2787, with permission. Part (c) adapted from Knöpfel T, Vranesic I, Staub C, Gähwiler BH (1991) Climbing fiber response in olivo-cerebellar slice cultures: II. Dynamics of cytosolic calcium in Purkinje cells. Eur. J. Neurophysiol.3, 343–348, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 12

Figure 16.11 Dendritic currents underlying the climbing fiber-evoked EPSP in Purkinje cells. (a) Modeling of the CF response recorded in current clamp mode in a dendrite (top trace) and the underlying [Ca2+]i transient (middle trace) and currents (I, bottom traces). The underlying currents are the synaptic glutamatergic current (Syn) which generates the climbing fiber EPSP, depolarizes the dendritic membrane and thus activates the dendritic P-type Ca2+ current (CaP) which further depolarizes the dendritic membrane, amplifies the EPSP and generates a Ca2+ spike (shown in top trace). The resultant [Ca2+]i increase (middle trace) activates the BK current (BK, bottom trace) which rapidly repolarizes the membrane. Adapted from De Schutter E, Bower JM (1994) An active membrane model of the cerebellar Purkinje cell: II. Simulation of synaptic response. J. Neurophysiol.71, 401–419, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 13

14 From Cellular and Molecular Neurophysiology, Fourth Edition. Figure 16.12 Activation of dendritic HVA Ca2+ channels by backpropagated Na+ action potential in hippocampal pyramidal neurons. Distal dendritic calcium influx is correlated with the efficacy of action potential backpropagation. (a) An action potential is evoked in the soma by a depolarizing current pulse. It backpropagates in the dendrites and is recorded at a dendritic site as a capacitative current at two different holding potentials (backpropagated AP). When the dendritic patch is held 20 mV more depolarized than resting potential (−45 mV), numerous openings of channels are observed following the action potential (arrow). In contrast, when the dendritic membrane is held at −105 mV, the backpropagated action potential does not evoke channel openings. (b) Spike-induced [Ca2+]i transients in a FURA-2 loaded neuron. Single action potentials (e.g. left) that propagate efficiently to the distal dendrite trigger robust calcium influx (expressed as the relative change in fluorescence, DF/F) in both proximal and distal dendritic compartments. Right: A different pyramidal neuron filled with Fura-2 exhibits weak action-potential backpropagation in the distal dendrites. The associated calcium influx shows significant attenuation in distal dendritic regions. Physiology scale bars: 20 mV, 1 ms. Imaging scale bars: 5% DF/F, 300 ms. Part (a) adapted from Magee JC, Johnston D (1995) Synaptic activation of voltage-gated channels in the dendrites of hippocampal pyramidal neurons. Science268, 301–304, with permission. Part (b) adapted from Golding NL, Kath WL, Spruston N (2001) Dichotomy of action-potential backpropagation in CA1 pyramidal neuron dendrites. J. Neurophysiol.86, 2998–3010, with permission. From Cellular and Molecular Neurophysiology, Fourth Edition. Copyright © 2015 Elsevier Ltd. All rights reserved. 14